| Ischemia and reperfusion (I/R) is defined as the restoration of blood perfusion after a period of blood supply interruption of the tissue/organ. Recovery of blood supply to the ischemic tissue/organ causes progressive lesion, which is named ischemia-reperfusion injury (IRI). Ischemia reperfusion injury refers to trauma, surgical operation and other diseases, in severe cases, reperfusion injury of skeletal muscles leads to systematic inflammation response syndrome and acute lung injury. Vacuum sealing drainage (VSD), is extensively used in surgery, this method of wound treatment reduces edema and inflammatory response, increases the blood perfusion and decreases the incidence of systematic inflammation response syndrome and multiply organ dysfunction. In the first part of this dissertation, we screened clinic cases of patients who suffered from open fractures of the lower limbs which combined with vascular injury, the aim of this part was to assess the therapeutic effect of vacuum sealing drainage on this group of patients. In the other part of this dissertation, we built a model of ischemia-reperfusion injury in rat hind limb, in order to investigate the protective effect as well as the mechanism of vacuum sealing drainage on skeletal muscle ischemia-reperfusion injury and associated lung injury.Part Ⅰ:clinical researchObjective In this part, a clinic study was designed to observe the effectiveness and safety of VSD applied to treat patients suffered from open fractures caused by crush injury and combined with vascular injury. Mehods Consecutive cases of patients with open fractures of the lower limbs accompanied with vascular injury were investigated. All patients included were allocated into VSD group or conventional group, during the treatment period after emergency debridement and vascular anastomosis, following information were compared between the two groups:ischemia time, mangled extremity severity score, the incidence of reexploration of the vessels, wound infection, ARDS, amputation and STSG/flaps, duration of wound close. The concentration of plasma CRP, IL-6 and myoglobin was compared either. Results There was no different of those parameters represent the severity of trauma between the two groups before the patients were undergoing medical treatment. Duration of wound close was shorten in VSD group compare with conventional group, but there was no difference with regard to the incidence of reexploration of the vessels, wound infection, ARDS, amputation and STSG/flaps. After 72 hours of treatment, patients in VSD group showed significantly lower concentration of plasma CRP, IL-6 and myoglobin. Conclusion In summary, vacuum sealing drainage promotes the healing after open fracture combined with vascular injury in lower limbs, and the incidence of complication such as rupture of the vessels, bleeding, vasospasm and thrombosis is equal to conventional wound treatment. But the influence of vacuum sealing drainage on skeletal muscle ischemia-reperfusion injury is in fact an area needs to be investigated.Part Ⅱ:experimental investigateObjective In this part, we build up an animal model to investigate the mechanism of vacuum sealing drainage which can alleviate skeletal muscle ischemia reperfusion injury and combined lung injury. Methods Male Sprague Dawley rats were selected to modeling ischemia-reperfusion injury in the hind limb, hemoclips were used to occlude and regain blood perfusion of left hind limbs of the rats, the time for occlusion was 4 hours and 8 hours for reperfusion. Three experiment groups were established, termed sham operation control (sham control group), ischemia-reperfusion injury (I/R group), ischemia and reperfusion accompanied with VSD treatment (I/R+VSD group),12 rats were assigned into each group. Half rats of each group were used for the test of pulmonary capillary permeability by Evans Blue leakage detection. At the end of the trial, samples of plasma, gastrocnemius and lung in the other half rats of each group were taken and stored at -80℃, used for the following tests: (1) Partial oxygen pressure of gastrocnemius was recorded by instrument for the measurement of Oxygen Partial Pressure. (2) Using a wet:dry ratio method to evaluate the oedema situation of the ischemic tissue. (3) MTT method was used for the determination of the skeletal muscle cell activity. (4) Oxidative stress indicators such as SOD (Superoxide Dismutase), MDA (Malondialdehyde), CAT (Catalase) and GSH (Glutathione) concentration levels in skeletal muscle tissue were determined by the commercial reagent kits. (5) The MPO (Myeloperoxidase) levels in skeletal muscle were also determined by commercial kit. (6) The VEGF (Vascular Endothelial Growth Factor) levels in skeletal muscle were determined by commercial ELISA kit. (7)Inflammatory cytokines IL-1β, IL-6, TNF-α concentrate in skeletal muscle, lung and serum were analysed by commercial ELISA kit. (8) Histopathology change in skeletal and lung tissue were evaluated by HE staining and HMGB1 (High Mobility Group Box-1 Protein) immunohistochemical staining were used to observe the morphologic changes and HMGB1 expression (qualitative and localization). Tunel were used for cell apoptosis assay. (9) The quantitative analysis for the HMGB1 in skeletal, lung and serum was detected at the gene (mRNA) expression level as well as the protein expression level. The statistic analysis was executed on SPSS 19. All datas were expressed as means and standard deviations. One-way analysis of variance (ANOVA) with the LSD (Equality of variance assumed) or Tamhane’s T2 (Equality of variance not assumed) post test was used to analyze the differences between the three groups. The results of partial oxygen pressure were analyzed by repeated measures analysis of variance. A P value of less than 0.05 was considered significant. Results (1) Rat pulmonary capillary leakage degree in I/R+VSD group was significantly lower than I/R group (t=5.289 P<0.001). (2) In the early stage of reperfusion VSD intervention could significantly decrease the tissue oxygen partial pressure when compared with the I/R group (P<0.05); the VSD device also maintained the pressure steady in the later period after reperfusion. (3) The wet/dry ratio of skeletal muscle and lung tissue in VSD group were all significantly lower than group I/R (t=7.718 P<0.001; r=4.427 P<0.001). (4) The results of MTT showed that the activity ratio of VSD skeletal muscle cells was significantly higher than that of I/R group (t=3.059 P=0.006). (5) The concentration of MAD in VSD group was significantly lower than group I/R (t=2.212 P=0.043), while the levels of GSH, CAT and SOD in VSD group were respectively significantly higher than those in I/R group (t=2.492 P=0.025; t=2.264 P=0.039; t=2.424 P=0.028). (6) The concentration of inflammation indicator, MPO in the VSD group was lower than the I/R group, and the difference was statistically significant (t=2.145 P=0.049). (7) The expression of VEGF in I/R group and VSD group was significantly higher than that in control group (F=77.616 P<0.001), and the difference between I/R group and VSD group was statistically significant (t=7.45 P<0.001). (8) Inflammatory cytokines IL-1β, IL-6 and TNF-α levels in group VSD were significantly lower than in group I/R(P<0.05). (9) Skeletal muscle HE stained sections showed the cell gap ratio and arrangement in VSD group was similar to the normal control group, however, in the I/R group, skeletal muscle cells showed increased widen for cell gap and the infiltration of inflammatory cells, and the injury scores in I/R group were significantly higher than VSD group (t=6.198 P<0.001); HE sections of lung tissue showed the degree of inflammatory cell infiltration, edema and acute exudation of VSD group were lower than I/R group, and the difference of lung injury score was significant between the two groups (t=6.033 P<0.001); immunohistochemical results showed the expression of inflammation associated protein in the VSD group was significantly lower than the I/R group; Tunel results indicated the apoptosis of skeletal muscles was observed after ischemia/reperfusion, but the VSD intervention could significantly reduce the proportion of the apoptosis by showing a significant difference compared with the I/R group (t=7.177 P<0.001). (10) The results of fluorescence quantitative PCR and Western blot indicated that the expression of HBGB1 in skeletal muscle as well as lung tissue of group I/R were all higher than group VSD, and the difference were statistically significant (P<0.05). The results of serum HMGB1 assayed by ELISA showed similar changes. Conclusion Vacuum sealing drainage can reduce oxygen partial pressure of skeletal muscle tissue and relieve the situation of oxidative stress. Vacuum sealing drainage can also reduce tissue edema, increase skeletal muscle perfusion as well as alleviate the "no reflow phenomenon", At last decrease skeletal muscle inflammation and reduce the concentration level of HMGB 1 protein, thereby reducing the acute lung injury followed by lower limb ischemia and reperfusion injury.In summary, the application of vacuum sealing drainage in patients with vascular injury associated with the open lower limb fractures can promote wound healing, reduce the systemic inflammatory response, to some extent, reduce the incidence of wound infection and acute lung injury, and bring benefits to the patients. Through the animal experiments, it can be concluded that VSD can reduce oxygen partial pressure of skeletal muscle tissue and reduce oxidative stress injury. VSD can also reduce tissue edema, increase skeletal muscle perfusion as well as alleviate the "no reflow phenomenon". At last, VSD can decrease skeletal muscle inflammation and reduce the concentration of HMGB1 protein, thereby reducing the acute lung injury followed by lower limb IRI. |
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